Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells
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Evaluation Summary:
The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in cultured T cell lines. In this study, the authors investigate the role of Fam49a and Fam49b in T cell development. The phenotype of mice lacking Fam49b mice suggests that it may be due to increased negative selection. The experiments are well performed and the results are convincing. Including data from TCR transgenic mice will solidify the view of the authors and greatly improve the impact of the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ + and CD8αα + TCRαβ + gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.
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Evaluation Summary:
The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in cultured T cell lines. In this study, the authors investigate the role of Fam49a and Fam49b in T cell development. The phenotype of mice lacking Fam49b mice suggests that it may be due to increased negative selection. The experiments are well performed and the results are convincing. Including data from TCR transgenic mice will solidify the view of the authors and greatly improve the impact of the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this study the authors investigate the role of Fam49a and Fam49b in T cell development. The phenotype of the Fam49b KO mice suggests that it may be due to increased negative selection. The experiments are well performed and the results are convincing; however, the study is not extensive and lacks a direct mechanistic element. Additional experiments are recommended to increase the impact of the study.
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Reviewer #2 (Public Review):
The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to thymic development of T cells is unknown. Since TCR signaling strength controls thymocyte development, Park et al. hypothesized that Fam49b could be critical for thymocyte development in vivo and investigated this by generating a novel knockout mouse line. They show that Fam49b is dispensable for positive selection but is required to prevent overly robust elimination of thymocytes at the negative selection stage, thereby identifying Fam49b as a critical regulator of negative selection. The conclusions of this paper are mostly well supported by data, but some aspects of …
Reviewer #2 (Public Review):
The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to thymic development of T cells is unknown. Since TCR signaling strength controls thymocyte development, Park et al. hypothesized that Fam49b could be critical for thymocyte development in vivo and investigated this by generating a novel knockout mouse line. They show that Fam49b is dispensable for positive selection but is required to prevent overly robust elimination of thymocytes at the negative selection stage, thereby identifying Fam49b as a critical regulator of negative selection. The conclusions of this paper are mostly well supported by data, but some aspects of TCR signaling and data analysis need to be clarified and extended. Besides, this study reveals a novel function of Fam49b as being required for optimal thymic development of lymphocytes and this could be relevant for T cell central tolerance and susceptibility to autoimmunity.
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Reviewer #3 (Public Review):
Using Crisp/Cas9 approaches, the authors generated Fam49a and Fam49b knockout mice. An overall analysis of T cells in Fam49a KO mice indicated no major alterations of thymic and peripheral T cell subsets and this strain was not further analyzed. In contrast, Fam49b knockout mice showed reduced numbers of SP thymocytes and peripheral T cells. A further analysis of Fam49b KO thymocytes subsets was performed and the authors analyzed the expression of markers such as TCRb, CD69, CD5, CCR7 as well as cleaved Caspase 3 expression. In addition, alterations in intestinal T cell subsets were observed. Based on the analysis, the authors conclude that Fam49b KO thymocytes undergo excessive negative selection and that Fam49b dampens TCR signal strength. The manuscript is well written and the data are clearly presented. …
Reviewer #3 (Public Review):
Using Crisp/Cas9 approaches, the authors generated Fam49a and Fam49b knockout mice. An overall analysis of T cells in Fam49a KO mice indicated no major alterations of thymic and peripheral T cell subsets and this strain was not further analyzed. In contrast, Fam49b knockout mice showed reduced numbers of SP thymocytes and peripheral T cells. A further analysis of Fam49b KO thymocytes subsets was performed and the authors analyzed the expression of markers such as TCRb, CD69, CD5, CCR7 as well as cleaved Caspase 3 expression. In addition, alterations in intestinal T cell subsets were observed. Based on the analysis, the authors conclude that Fam49b KO thymocytes undergo excessive negative selection and that Fam49b dampens TCR signal strength. The manuscript is well written and the data are clearly presented. However, the conclusion that there is excessive negative selection should be strengthened by the inclusion of additional experiments.
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